Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicemia, and meningitis. In recent years, it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work, we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types, including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by transcriptome sequencing (RNAseq), confirms that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly, we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS, and SPD_0921 are not involved in spnIII recombination, suggesting that a currently unknown mechanism is responsible for the recombination of these phase-variable type I systems.IMPORTANCEStreptococcus pneumoniae is a leading cause of pneumonia, septicemia, and meningitis. The discovery that genetic rearrangements in a type I restriction-modification locus can impact gene regulation and colony morphology led to a new understanding of how this pathogen switches from harmless colonizer to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction-modification enzyme, occur across many different pneumococcal serotypes and sequence types and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site-specific recombination.

Persistent Staphylococcus aureus isolates from two independent cases of bacteremia display increased bacterial fitness and novel immune evasion phenotypes.

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

Virulence of enteropathogenic Escherichia coli, a global pathogen.

Enteropathogenic Escherichia coli (EPEC) remains an important cause of diarrheal disease worldwide. Research into EPEC is intense and provides a good virulence model of other E. coli infections as well as other pathogenic bacteria. Although the virulence mechanisms are now better understood, they are extremely complex and much remains to be learnt. The pathogenesis of EPEC depends on the formation of an ultrastructural lesion in which the bacteria make intimate contact with the host apical enterocyte membrane. The formation of this lesion is a consequence of the ability of EPEC to adhere in a localized manner to the host cell, aided by bundle-forming pili. Tyrosine phosphorylation and signal transduction events occur within the host cell at the lesion site, leading to a disruption of the host cell mechanisms and, consequently, to diarrhea. These result from the action of highly regulated EPEC secreted proteins which are released via a type III secretion system, many genes of which are located within a pathogenicity island known as the locus of enterocyte effacement. Over the last few years, dramatic increases in our knowledge of EPEC virulence have taken place. This review therefore aims to provide a broad overview of and update to the virulence aspects of EPEC.

Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer.

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.

Enteropathogenic Escherichia coli infection: history and clinical aspects.

Diarrhoeagenic Escherichia coli remains an important cause of diarrhoeal disease worldwide. In terms of global public health, enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli are the most important. However, enterohaemorrhagic E. coli has emerged as a cause of disease in developed countries in recent years, and a number of large outbreaks have been reported. Therefore, the importance of research into diarrhoeagenic E. coli remains an important issue. EPEC is the most widespread of the diarrhoeagenic E. coli and provides a good virulence model for other E. coli infections, as well as other pathogenic bacteria. Although the virulence mechanisms of E. coli are now better understood, there remains much to be learned before effective treatments can be developed. Type III secretion mechanisms, the locus of enterocyte effacement and various toxins are all involved in the pathogenesis of the various diarrhoeagenic E. coli and may provide targets for future therapies. This review aims to provide an update on the worldwide problem of diarrhoeagenic E. coli by focusing on EPEC, and describes the history of the organism, its incidence and the clinical aspects of infection.

Catecholamine inotropes as growth factors for Staphylococcus epidermidis and other coagulase-negative staphylococci.

Drugs commonly used in intensive care settings were assayed for their ability to affect the growth of Staphylococcus epidermidis in a minimal salts medium containing 30% serum. Of 28 compounds tested, the inotropic catecholamines adrenaline, dobutamine, dopamine, isoprenaline and noradrenaline significantly stimulated bacterial growth. These drugs, but not structurally similar compounds lacking a dihydroxybenzoyl moiety (such as tyramine, phenylephrine and salbutamol), were able to remove iron from iron-saturated transferrin and to supply transferrin-bound 55Fe to S. epidermidis cells. Similar results were observed with a range of coagulase-negative staphylococci associated with line infections, but not with Staphylococcus aureus (including MRSA).

The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin.

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [(3)H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.

Stimulation of bacterial growth by heat-stable, norepinephrine-induced autoinducers.

The ability of norepinephrine to increase the growth of Escherichia coli in a serum-based medium has previously been shown to be due to the production of an autoinducer of growth during early log phase. Seventeen Gram-negative and 6 Gram-positive clinical isolates were examined for a similar ability to respond to norepinephrine, and to synthesise autoinducer. The majority of Gram-negative strains both produced and responded to heat-stable norepinephrine-induced autoinducers of growth. Most of these autoinducers showed a high degree of cross-species activity, suggesting the existence of a novel family of Gram-negative bacterial signalling molecules. In contrast, although a number of Gram-positive strains were able to respond to norepinephrine, the majority failed to produce autoinducers in the presence of norepinephrine.